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*
PharMingen, San Diego, CA 92121;
Immunobiology Section, Yale University School of Medicine, New Haven, CT 06510;
Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029;
§
Korea National University, Seoul, Republic of Korea; and
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Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The apoptotic machinery has a complicated and poorly understood relationship to cell cycle control. In T cells, activation is essential to induce full susceptibility to CD95-induced apoptosis, yet apoptotic signals from CD95 preferentially kill thymocytes or activated T cells that are in the G0/G1 phases of the cycle, but spare cells in S phase (10). This tight linkage between CD95-induced apoptosis and the G1 phase of the cell cycle prompted us to investigate whether CD95 signals were aberrantly activating the G1/S cell cycle checkpoint.
In this report, we show that CD95 ligation causes the induction of the protein p21cip-1/WAF-1. This protein is a cell cycle regulator which is involved in the G1/S and G2/M checkpoints, and is induced in response to DNA damage through the action of the tumor suppressor protein, p53. Growth factors may cause G1 arrest in some cells by p53-independent p21cip-1/WAF-1 up-regulation. The nerve growth factor receptor shares strong homology with the CD95 molecule. This receptor induces differentiation through the ERK kinase-dependent up-regulation of p21cip-1/WAF-1 and also inhibits cyclin-dependent kinase (cdk) activity, causing cell cycle arrest (11). The best-understood action of p21cip-1/WAF-1 is the inhibition of cdks, leading to dephosphorylation of the pocket proteins p107 and p110Rb (12, 13). Typically, this results in cell cycle arrest during the G1 phase. In our experiments, the p21cip-1/WAF-1 signal potentiated CD95-induced apoptosis of T cells because apoptosis was reduced in p21cip-1/WAF-1-deficient T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The experiments used T cells from C57BL6 mice (The Jackson Laboratory, Bar Harbor, ME), unless otherwise stated. B6.Faslpr mice were obtained from Dr. Mark Shlomchik (Yale University, New Haven, CT). The Faslpr-cg mice were derived from Dr. A. Matsuzawa’s colony (University of Tokyo, Tokyo, Japan) and bred at Yale, whereas matched normal controls for these mice were obtained from The Jackson Laboratory. The p53-deficient (p53-/-) mice and appropriate matched control mice were from Taconic Farms (Germantown, NY). The p21cip-1/WAF-1-deficient (p21-/-) mice were obtained from Dr. Phillip Leder (Harvard University, Cambridge, MA). Animals were maintained and bred by the Yale Animal Resources Center and used at between 6 and 8 wk of age.
Materials
Polyclonal rabbit Ab to p21cip-1/WAF-1 (C-19) and goat anti-rabbit Ig antiserum coupled to HRP were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin Ab was from Sigma (St. Louis, MO). ZVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and DEVD-CHO (succinyl-Asp-Glu-Val-Asp-aldehyde) were from Calbiochem (La Jolla, CA). Other Abs were obtained from PharMingen (San Diego, CA). Con A and Histopaque solutions (of density 1.119 g/ml and 1.07 g/ml) were from Sigma (St. Louis, MO). Tissue culture medium was RPMI 1640 (prepared in-house by the Yale Immunobiology media facility) supplemented with 5% FCS, 2 mM L-glutamine, 50 µM 2-ME, 10 mM HEPES, and 100 U/ml each of penicillin and streptomycin.
Cell culture
Thymocytes were isolated by mechanical disruption of fresh mouse
thymi in a ground-glass tissue grinder, washed two times in serum-free
RPMI 1640 medium, and used ex vivo. Killing experiments were cocultures
between fresh thymocytes or spleen T cell blasts, and control or
CD95L-transfected fibroblasts. Blasts were isolated from spleen cells
cultured for 3 days with 2 µg/ml of Con A, or 2 µg/ml of
anti-CD3 Ab (145-2C11). The fibroblasts were NIH 3T3 cells,
transfected either with empty vector (pSR
72.N1) for the control, or
the same vector containing a murine CD95L cDNA (14). Both
fibroblast lines were maintained under constant selection using G418,
and plated without G418 only to establish semiconfluent monolayers for
the experiments. To test CD95L-induced killing and
p21cip-1/WAF-1 induction, thymocytes or T cell
blasts were suspended at 1 x 107 cells in a
final volume 10 ml of RPMI culture medium and layered over control or
CD95L-transfected fibroblasts in 10 cm culture dishes (Falcon 35-3003,
Becton Dickinson, Franklin Lakes, NJ). The T cells did not form
adhesions to the fibroblasts and were easily recovered after various
time intervals by rocking of the tissue culture dishes, followed by
gentle aspiration with a Pasteur pipette. As an alternative to the
coculture, T cell blasts were cultured with 0.5 µg/ml of Jo-2
anti-CD95 Ab, followed after 5 min by the addition of polyclonal
anti-mouse Ig at 1 µg/ml. Such cross-linked cells were analyzed
after 24 h. Control cells were subjected to the cross-linking step
only. Recovered cells were counted, identifying viable cells by trypan
blue exclusion. Cells were stained with propidium iodide (PI; from
Sigma) to determine DNA content and, thus, to detect subdiploid cells
that were undergoing apoptosis (15). A suspension of cells
was incubated for 45 min in PBS containing 50 µg/ml of PI, 0.1%
sodium citrate, 0.3% Nonidet P-40, and 50 µg/ml RNase (Sigma).
PI-stained cells were examined using a FACScan (Becton Dickinson,
Mountain View, CA), and data were analyzed using CellQuest
software.
Immunoblotting
Thymocytes or T cell blasts were centrifuged, and the pellet lysed in the RIPA lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mg/ml PMSF). Total cell extracts were normalized for protein concentration, and samples containing 20 µg of protein were resolved on 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) Immobilon membranes (Millipore, Bedford, MA) using an electrophoretic transfer apparatus from Bio-Rad (Hercules, CA). Transfer was typically for 1 h at 100 V in 27.5 mM Tris/0.2 M glycine buffer. After transfer, membranes were blocked for a minimum of 1hr with 5% dry milk in 50 mM Tris-buffered saline (pH 7.5) containing 0.05% Tween 20. Blots were probed overnight with rabbit Ab to p21cip-1/WAF-1, or mouse anti-actin as a loading control. After extensive washing, proteins were tagged using goat anti-rabbit HRP, and then visualized using the Enhanced Chemiluminescent kit from Amersham Life Sciences (Buckinghamshire, U.K.).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Short-term coculture of thymocytes with CD95L-expressing
fibroblasts resulted in rapid apoptosis accompanied by induction of
p21cip-1/WAF-1. Thymocytes were cocultured for
various time periods between 3 and 5 h with a monolayer of control
(vector-transfected) or cytotoxic (CD95L-transfected) 3T3 cells, which
resulted in apoptosis, detected by loss of viable cells based on trypan
blue exclusion. Lymphocytes were harvested and examined for
p21cip-1/WAF-1 expression by Western blotting. In
normal mouse thymocytes, induction was apparent in 3 h after the
start of coculture (Fig. 1
B).
We also observed p21cip-1/WAF-1 induction in Con
A-activated spleen cell blasts and in the Jurkat human T cell line
(data not shown), but not in thymocytes from
Faslpr mice (Fig. 1B). This suggests
that CD95-mediated induction of p21cip-1/WAF-1 is
a general property of CD95-expressing T cells.
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CD95-induced up-regulation of p21cip-1/WAF-1 is inhibited by caspase inhibitors
Caspases may be inhibited by short peptides with homology
for the caspase target site. Such inhibitors have specificity for
subsets of caspases, and can inhibit CD95-induced cleavage of caspase
substrates and apoptosis of target cells (19, 20). To
analyze the relationship between the caspase family and CD95-induced
p21cip-1/WAF-1 induction, we used two kinds
of peptide inhibitors with different specificities. Z-VAD-fmk, an
inhibitor for a broad spectrum of caspases, blocked the
CD95-induced up-regulation of p21cip-1/WAF-1
almost completely (Fig. 2B,
compare lanes 3 and 4). Normal mouse thymocytes
were killed by CD95L-transfected 3T3 cells, and the killing was
inhibited by the addition of 50 µM ZVAD-fmk during the coculture
(Fig. 2A). In contrast, DEVD-CHO, a specific inhibitor for
caspase 3, blocked killing by 50%, but did not block
p21cip-1/WAF-1 induction in response to CD95
ligation. In the experiment shown, the
p21cip-1/WAF-1 protein level in fact appeared to
increase in the presence of DEVD-CHO.
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below). Second, if p21cip-1/WAF-1
induction were a downstream effect of cell death, there is no reason
why cell death would be reduced in p21-cip-1 deficient thymocytes.
However, we found that it is reduced (see Fig. 4 below). We conclude
that the induction of p21cip-1/WAF-1 is most
likely to occur in parallel with apoptosis, rather than as a
consequence of it.
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B, may be significant.
Such induction is a consistent finding and was observed in three
similar experiments. We think it occurs because the caspase-3 inhibitor
is preventing apoptosis of some of those cells in which CD95 signaling
results in p21cip-1/WAF-1 being induced. We are cautious about attributing too much significance to the differential effects of ZVAD-fmk and DEVD-CHO. The fact that these two agents have different effect on p21cip-1/WAF-1 induction (where ZVAD-fmk is a complete inhibitor, while DEVD-fmk has no inhibitory effect) and cell death (where both inhibit, but to a different degrees) might simply be due to their different biological potency. However, it is also possible that p21cip-1/WAF-1 induction depends on caspase-8, but not on caspase-3. This possibility is in accordance with the observation that CD95-mediated activation of c-Jun N-terminal kinase (JNK) and p38 is cell death-dependent, but caspase 3-independent (7, 8). The data are consistent with the idea that these two effects of CD95 signaling both depend on upstream, but not effector caspases.
To verify that the induction of p21cip-1/WAF-1
was due to recruitment of the adaptor molecules that activate
caspase-8, experiments were conducted in cells from
Faslpr-cg mice. In these mice, a point mutation
in the DD of CD95 results in failure to recruit the adaptor molecule
FADD. This mutation therefore prevents caspase-8 activation, and
CD95-mediated apoptosis (21). In line with expectations,
thymocytes from Faslpr-cg mice were not killed
by coculture with CD95L-transfected 3T3 cells (Fig. 2A) and
p21cip-1/WAF-1 was not induced (Fig. 2B). We conclude that the upstream components of the
DD-FADD-caspase pathway are required for CD95-mediated
p21cip-1/WAF-1 induction.
p21cip-1/WAF-1 induction is p53-independent
To determine whether CD95 acts on
p21cip-1/WAF-1 via p53, thymocytes from control
or p53-deficient (p53-/-) mice were cocultured
with control or CD95L-transfected fibroblasts. Cells were harvested for
analysis by PI staining and Western blotting. The
p53-/- cells were effectively killed by
CD95L-transfected fibroblasts, showing that CD95-induced apoptosis was
not p53-dependent (Fig. 3B).
Furthermore, although the basal level of
p21cip-1/WAF-1 was extremely low in
p53-/- thymocytes, there was clear induction of
p21cip-1/WAF-1 by coculture with
CD95L-transfected fibroblasts (Fig. 3C). We
conclude that the induction of p21cip-1/WAF-1 by
CD95 is fully independent of p53. This mechanism of
p21cip-1/WAF-1 induction is unusual, but not
unique. A number of growth factors, including those in normal serum
that cause the proliferation of fibroblasts, cause induction of
p21cip-1/WAF-1 by a p53-independent pathway
(22).
Function of CD95-mediated p21cip-1/WAF-1 induction
In some cell types, p21cip-1-WAF-1 induction is associated with G1 arrest. However, in T-lineage cells the relationship among CD95, apoptosis, cell cycle status, p21cip-1/WAF-1, and caspases is a complex one. Freshly isolated ex vivo thymocytes contain around 10% of cells in S phase that may be labeled with 5-bromo-2'-deoxyuridine (BrdU) in short-term culture as we have shown before (10). Although thymocytes after exposure to CD95L were enriched in S phase cells, this was not due to cell cycle progression but simply to the selective killing of G0/G1 cells (10). When caspases are completely blocked, for example by ZVAD-fmk, there is no such death and no selective cell loss. If the p21cip-1/WAF-1 induction had been independent of caspase activation, we would have expected that the induction of p21cip-1/WAF-1 would lead to inhibition of cdk-2/cyclin complexes, dephosphorylation of p107 and p110Rb, and cell cycle arrest. However, when T cells or thymocytes were exposed to CD95L in the presence of ZVAD-fmk plus BrdU, we found no evidence of CD95-induced G1 arrest in T cells. There was neither a decrease in cells in S/G2/M phases of the cycle, based on a determination of the DNA content by PI staining, nor a decrease in cells in S based on BrdU incorporation, detected using an anti-BrdU Ab to stain permeabilized cells (data not shown).
Next we considered the possibility that CD95-induced p21cip-1/WAF-1 up-regulation might be the molecular basis for the costimulatory effect of CD95 reported in human T cells (4). In these experiments, limiting concentrations of anti-CD3 were used to activate T cells, either co-immobilized with anti-CD95 Ab on plastic, or with normal or CD95L-deficient (FasLgld) spleen cells as APC. If we have been able to detect any enhanced proliferation of naive T cells in the presence of CD95 ligation, we would have been able to test the importance of p21cip-1/WAF-1 using p21cip-1/WAF-1-deficient T cells. However, we were not able to show reproducible costimulatory effects of CD95L on mouse T cells, and the presence or absence of p21cip-1/WAF-1 in the T cells made no obvious difference to the proliferation in either case. We therefore found no evidence for CD95 costimulation mediated by p21cip-1/WAF-1 (data not shown).
We tested whether the p21cip-1/WAF-1 mediated
signal would regulate the effects of caspase signaling. It has already
been shown that truncation of the exon 9-encoded C terminus of CD95
results in enhanced apoptosis, suggesting the presence of a regulatory
signaling pathway originating from this part of the molecule, distal to
the DD (23). This establishes the point that CD95 signals
are complex, and that signals transmitted via different pathways
may interact to regulate the caspase-mediated death signal. However,
our data show that the signal that results in the induction of
p21cip-1/WAF-1 originates from the DD and
involves the activation of at least upstream caspases. We tested the
possibility that such induction of p21cip-1/WAF-1
might inhibit caspase-mediated apoptosis in a manner analogous to the
signal from the C terminus. If CD95 were to transmit an
anti-apoptotic signal via p21cip-1/WAF-1, it
would be expected that CD95 signaling in
p21cip-1/WAF-1 deficient
(p21-/-) cells would be enhanced. However, we
observed the opposite effect. Thymocytes from normal or
p21-/- mice were cocultured with control or
CD95L-transfected 3T3 fibroblasts; and, at various time intervals
between 1 and 5 h, lymphocytes were harvested for analysis by PI
staining. At 3 and 5 h, there was effective killing of normal
thymocytes resulting in the accumulation of subdiploid cells, but there
was around 50% less killing of p21-/-
thymocytes (Fig. 4). This difference in
killing was highly significant (p < 0.02 at
3 h and p < 0.001 at 5 h; significance was
evaluated using a t test, n = 5
experiments). The result was not simply due to a difference in CD95
expression, because staining of p21-/-
thymocytes showed a level of CD95 expression that was identical to
normal B6 thymocytes. In addition to these experiments in thymocytes,
diminished CD95-induced apoptosis was seen in
p21-/- spleen T cell blasts cocultured with
CD95L-expressing 3T3 cells, and in p21-/-
thymocytes treated with the anti-CD95 Ab Jo-2 plus a cross-linking
second Ab (data not shown). Spleen cells of
p21-/- mice also express CD95 at the same level
as normal control cells. The Ab was used as an alternative ligand for
CD95 on the T cell surface to exclude the possibility that the effects
of p21cip-1/WAF-1 in CD95-induced apoptosis were
mediated through other cell surface molecules. The similar effects of
CD95L expressed on 3T3 cells and the cross-linked anti-CD95 Ab
argue against this possibility.
In summary, CD95-induced expression of p21cip-1/WAF-1 is independent of p53, but depends on caspases. These results suggest that p21cip-1/WAF-1 signals originate from the DD, and not the C-terminal regulatory domain of CD95. The function of p21cip-1/WAF-1 in CD95-mediated apoptosis appears to be to promote killing of the target cell.
Biological significance
The participation of p21cip-1/WAF-1 in p53-triggered cell death is still not clear. Some authors have shown that induction of apoptosis by p53 is associated with the up-regulation of endogenous p21cip-1/WAF-1 (24) and Bax protein (25), but others have shown that the p21cip-1/WAF-1 gene is not essential for apoptosis (26). One set of experiments used the established cell line BHK21 expressing human p21cip-1/WAF-1 conditionally under a Tetracycline (Tet)-repressible promoter. In these cell lines, removal of Tet resulted in about a 10-fold increase in p21cip-1/WAF-1 expression, slower growth, cell-cycle arrest, and an increase in cell death (27).
There are two classes of CDK inhibitors, the INK4 inhibitors (INK4a to INK4d), which act specifically on cyclin D-dependent kinases, and the Cip/kip family (p21cip-1/WAF-1, p27kip1, and p57kip2), which function broadly to inhibit cyclins E and A as well as cyclin B-dependent kinases. The best-understood function of p21cip-1/WAF-1 is to bind to and inhibit cyclin-dependent kinases, principally cdk-4 and cdk-6 (12, 13, 28). These kinases are believed to act by phosphorylating the pocket proteins p130, p107, and p110Rb, which abrogates their inhibitory function and allows the activation of the transcription factors to which they bind, namely the E2F proteins (29). The E2F family is vitally important in G1 progression and the induction of S phase, but also has a poorly understood role in the induction of apoptosis (30, 31, 32, 33, 34). The induction of p21cip-1/WAF-1 by CD95 signaling could result in cdk-4 and cdk-6 inhibition, leading to pocket protein de-phosphorylation and changes in E2F function. One possibility is that in the presence of active caspases, cdk inhibition results in the repression of the cell cycle progression but not the apoptotic effect of E2F, two functions that appear to be separable (33). However, a role for E2F in CD95-induced apoptosis remains highly speculative.
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Acknowledgments |
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Footnotes |
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2 R.H. and B.B. contributed equally to the work.
3 Address correspondence and reprint requests to Dr. I. N. Crispe, Section of Immunobiology, Yale School of Medicine, 310 Cedar Street, P.O. Box 208011, BML 462, New Haven, CT 06520-8011. E-mail address:
4 Abbreviations used in this paper: CD95L, CD95 ligand; FADD, Fas-associated death domain; DD, death domain; cdk, cyclin-dependent kinase; ZVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; DEVD-CHO, succinyl-Asp-Glu-Val-Asp-aldehyde; PI, propidium iodide.
Received for publication August 9, 1999. Accepted for publication February 3, 2000.
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) with
CD95L-transfected fibroblasts (
); p21-/- thymocytes
were cocultured with control fibroblasts (•) with CD95L-transfected
fibroblasts (
). The experiment shown is one of five that gave
similar results.